The present invention relates to cytotoxic T-cell (CTL) epitopes within Epstein-Barr virus. The present invention also relates to subunit vaccines including CTL epitopes.
Epstein-Barr virus (EBV) is a gamma herpesvirus which establishes a latent lifelong infection in the host following acute infection (14,15). While primary infection generally occurs in childhood without significant morbidity, adolescents and young adults may present with the symptoms of acute infectious mononucleosis (IM). The main feature of IM is a self-limiting lymphoproliferation involving both T and B cells accompanied by clinical symptoms such as fever and lymphadenopathy (52,53). Occasionally, the clinical symptoms persist and recur for extended periods after the initial infection. Episodic IM such as this has been described as chronic active EBV infection or, in some cases, severe chronic active EBV infection (35). EBV DNA has been detected in both serum and peripheral blood lymphocytes (PBL) during acute IM with the levels of detectable DNA gradually decreasing as the illness abates (21.22.58).
Evidence for latent EBV infection includes the observation that spontaneous lymphoblastoid cell lines (LCLs), expressing latent proteins, can be regularly established from healthy immune individuals after explantation of either lymph node tissue (34) or fractionated B lymphocytes (59). Although latent EBV infection is usually asymptomatic, sequential studies have established that recrudescence of viral replication in the oral cavity may result in release of infectious virus (59). The exact site of persistence of the virus is uncertain, but the available evidence suggests that small lymphocytes in the circulation harbour the virus in a nonproductive episomal state (24). Accordingly, in asymptomatic donors, EBV DNA is detectable by sensitive PCR analysis in PBL expressing the B-cell marker CD19(29,55).
EBV is also involved in post transplant lymphoproliferative disease, which involves a polyclonal expansion of EBV infected B-cells which is a life threatening lymphoma especially in transplantation patients. EBV is also involved in nasopharyngeal carcinoma and Hodgkinson""s disease.
Two types of EBV (types A and B or types 1 and 2) are distinguishable primarily on the basis of variation in the DNA and protein sequences of the latent EBV-associated nuclear antigens (referred to here as EBNAs-2A, -3A, -4A and -6A from the type A virus and EBNAs-2B, -3B, -4B and -6B from the type B virus) (9,46,48). Sequencing of the prototypical isolates of type A and type B EBV (B95-8 and Ag-876 respectively) in these regions revealed 53% amino acid homology between EBNA-2A and EBNA-2B (9) and 72-84% homology between EBNAs-3A and -3B. EBNAs-4A and -4B, and EBNAs-6A and -6B (46). Strain variation due to other DNA alterations or deletions as well as these A/B type differences have been defined at the protein (12) and the DNA level (16,25,26) and recombination between multiple infecting strains was found to occur frequently in oral hairy leukoplakia lesions (56). These variations offer an alternative means of categorising EBV isolates but the primary distinction of type A and type B is still useful. Type A EBV is more readily isolated from healthy donors: type B EBV infections or dual infections with both type A and type B have proven easier to detect in immunosuppressed or HIV infected individuals (5,47,51). A higher incidence of type B infection in some studies led to the suggestion that type B or dual infections are, in fact, relatively common and that resident type B virus levels increase during immunosuppression (3,20,51).
It appears that latent EBV infection is primarily controlled by HLA class I-restricted memory cytotoxic T cell (CTL) responses (reviewed in (18)). These CTL responses can be reactivated in vitro by stimulating lymphocytes from seropositive individuals with autologous lymphoblastoid cell lines (LCLs) which express and present MHC class I and class II restricted epitopes at the cell surface. Several of these epitopes have been identified using target cells infected by recombinant vaccinia constructs (17,19,32,33). Epitopes specific for type A EBV as well as cross-reactive epitopes encoded by both types A and B EBV have been defined (18) but no epitopes specific for type B EBV have been reported thus far. In addition co-pending International Patent Application No. WO 95/001400, the disclosure of which is incorporated herein by cross reference discloses a number of EBV CTL epitopes. In the present study, the response of a donor exposed to both type A and type B EBV was investigated and an epitope specific for type B EBV as well as a new cross-reactive epitope were identified.
In a first aspect the present invention consists in a cytotoxic Epstein-Barr virus T-cell epitope, the epitope being selected from the group consisting of QVKWRMTTL (SEQ ID NO: 31), VFSDGRVAC (SEQ ID NO: 32), VPAPAGPIV (SEQ ID NO: 33), TYSAGIVQI (SEQ ID NO: 34), LLDFVRFMGV (SEQ ID NO: 35), QNGALAINTF (SEQ ID NO: 36), VSSDGRVAC (SEQ ID NO: 37), VSSEGRVAC (SEQ ID NO: 38), VSSDGRVPC (SEQ ID NO: 39), VSSDGLVAC (SEQ ID NO: 40), VSSDGQVAC (SEQ ID NO: 41), VSSDGRVVC (SEQ ID NO: 42), VPAPPVGPIV (SEQ ID NO: 43), VEITPYEPTG (SEQ ID NO: 44), VEITPYEPTW (SEQ ID NO: 45), VELTPYKPTW (SEQ ID NO: 46), RRIYDLIKL (SEQ ID NO: 47), RKIYDLIEL (SEQ ID NO: 48) and PYLFWLAGI (SEQ ID NO: 49).
In a second aspect the present invention consists in a subunit vaccine, the vaccine including at least one T-cell epitope selected from the group consisting of QVKWRMTTL (SEQ ID NO: 31), VFSDGRVAC (SEQ ID NO: 32), VPAPAGPIV (SEQ ID NO: 33), TYSAGIVQI (SEQ ID NO: 34), LLDFVRFMGV (SEQ ID NO: 35), QNGALAINTF (SEQ ID NO: 36), VSSDGRVAC (SEQ ID NO: 37), VSSEGRVAC (SEQ ID NO: 38), VSSDGRVPC (SEQ ID NO: 39), VSSDGLVAC (SEQ ID NO: 40), VSSDGQVAC (SEQ ID NO: 41), VSSDGRVVC (SEQ ID NO: 42), VPAPPVGPIV (SEQ ID NO: 43), VEITPYEPTG (SEQ ID NO: 44), VEITPYEPTW (SEQ ID NO: 45), VELTPYKPTW (SEQ ID NO: 46), RRIYDLIKL (SEQ ID NO: 47), RKIYDLIEL (SEQ ID NO: 48) and PYLFWLAGI (SEQ ID NO: 49).
In a preferred embodiment of this aspect of the present invention the subunit vaccine includes at least one further epitope selected from the group consisting of TSLYNLRRGTALA (SEQ ID NO: 1), DTPLIPLTIF (SEQ ID NO: 2), TVFYNIPPMPL (SEQ ID NO: 3), VEITPYKPTW (SEQ ID NO: 4), VSFIEFVGW (SEQ ID NO: 5), FRKAQIQGL (SEQ ID NO: 6), FLRGRAYGL (SEQ ID NO:7), QAKWRLQTL (SEQ ID NO: 8), SVRDRLARL (SEQ ID NO: 9), YPLHEQHGM (SEQ ID NO: 10), HLAAQGMAY (SEQ ID NO: 11), RPPIFIRRL (SEQ ID NO: 12), RLRAEAGVK (SEQ ID NO: 13), IVTDFSVIK (SEQ ID NO: 14), AVFDRKSDAK (SEQ ID NO: 15), NPTQAPVIQLVHAVY (SEQ ID NO: 16), LPGPQVTAVLLHEES (SEQ ID NO: 17), DEPASTEPVHDQLL (SEQ ID NO: 18), RYSIFFDY (SEQ ID NO: 19), AVLLHEESM (SEQ ID NO: 20), RRARSLSAERY (SEQ ID NO: 21), EENLLDFVRF (SEQ ID NO: 22), KEHVIQNAF (SEQ ID NO: 23), RRIYDLIEL (SEQ ID NO: 24), QPRAPIRPI (SEQ ID NO: 25), EGGVGWRHW (SEQ ID NO: 26), CLGGLLTMV (SEQ ID NO: 27), RRRWRRLTV (SEQ ID NO: 28), RAKFKQLL (SEQ ID NO: 29) and RKCCRAKFKQLLQHYR (SEQ ID NO: 30).
In a further preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31) and FLRGRAYGL (SEQ ID NO:7). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 63.7% of the caucasian population.
In a yet further preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7) and EENLLDFVRF (SEQ ID NO: 22). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 71.1% of the caucasian population.
In another preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7) and QPRAPIRPI (SEQ ID NO: 25). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 74.1% of the caucasian population.
In a still further preferred embodiment of this aspect of the present invention the subunit vaccine includes the cytotoxic T-cell epitopes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7), EENLLDFVRF (SEQ ID NO: 22) and QPRAPIRPI (SEQ ID NO: 25). An analysis of allele frequency in the HLA listings in the Queensland Institute of Medical Research data base shows that a vaccine including these epitopes would provide protection for 81.5% of the caucasian population. Given the fact that about 50% of all individuals that are not covered by vaccination will become EBV positive without any symptoms, the combination of epitopes listed above will result in a vaccine with more than 90% efficacy. This is of high commercial value.
In a further preferred form of the present invention the vaccine comprises a water-in-oil formulation. It is further preferred that the vaccine includes at least one antigen to which the individual will mount an anamnestic response in addition to the at least one cytotoxic T-cell epitope.
The at least one antigen is preferably selected from the group consisting of tetanus toxoid, diphtheria toxoid, Bordetella pertussis antigens, poliovirus antigens, purified protein derivative (PPD), gp350 protein (Thorley-Lawson, D. A. and Poodry, C. A. (1982). Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo. J. Virol. 43, 730-736), helper epitopes and combinations thereof and is preferably tetanus toxoid.
It is preferred that the water-in-oil formulation is Montanide ISA 720. Additional information regarding this formulation can be found in WO 95/24926, the disclosure of which is incorporated herein by cross reference.
The subunit vaccine may also be formulated using ISCOMs. Further information regarding ISCOMs can be found in Australian Patent Nos. 558258, 590904, 632067, 589915, the disclosures of which are included herein by cross reference.
In a third aspect the present invention consists in a nucleic acid vaccine, the vaccine including a nucleic acid sequence encoding at least one of the cytotoxic T-cell epitopes of the first aspect of the present invention and optionally at least one cytotoxic T-cell epitope selected from the group consisting of TSLYNLRRGTALA (SEQ ID NO: 1), DTPLIPLTIF (SEQ ID NO: 2), TVFYNIPPMPL (SEQ ID NO: 3), VEITPYKPTW (SEQ ID NO: 4), VSFIEFVGW (SEQ ID NO: 5), FRKAQIQGL (SEQ ID NO: 6), FLRGRAYGL (SEQ ID NO:7), QAKWRLQTL (SEQ ID NO: 8), SVRDRLARL (SEQ ID NO: 9), YPLHEQHGM (SEQ ID NO: 10), HLAAQGMAY (SEQ ID NO: 11), RPPIFIRRL (SEQ ID NO: 12), RLRAEAGVK (SEQ ID NO: 13), IVTDFSVIK (SEQ ID NO: 14), AVFDRKSDAK (SEQ ID NO: 15), NPTQAPVIQLVHAVY (SEQ ID NO: 16), LPGPQVTAVLLHEES (SEQ ID NO: 17), DEPASTEPVHDQLL (SEQ ID NO: 18), RYSIFFDY (SEQ ID NO: 19), AVLLHEESM (SEQ ID NO: 20), RRARSLSAERY (SEQ ID NO: 21), EENLLDFVRF (SEQ ID NO: 22), KEHVIQNAF (SEQ ID NO: 23), RRIYDLIEL (SEQ ID NO: 24), QPRAPIRPI (SEQ ID NO: 25), EGGVGWRHW (SEQ ID NO: 26), CLGGLLTMV (SEQ ID NO: 27), RRRWRRLTV (SEQ ID NO: 28), RAKFKQLL (SEQ ID NO: 29) and RKCCRAKFKQLLQHYR (SEQ ID NO: 30).
Further information regarding nucleic acid vaccines can be found in WO 96/03144 the disclosure of which is incorporated herein by cross reference. As will be appreciated by those skilled in the field the DNA can be delivered using a suitable viral or bacterial vector.
In a preferred embodiment of this aspect of the present invention the nucleic acid sequence encodes LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31) and (FLRGRAYGL (SEQ ID NO:7), or LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7) and EENLLDFVRF (SEQ ID NO: 22), or LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7) and QPRAPIRPI (SEQ ID NO: 25), or LLDFVRFMGV (SEQ ID NO: 35), QVKWRMTTL (SEQ ID NO: 31), FLRGRAYGL (SEQ ID NO:7), EENLLDFVRF (SEQ ID NO: 22) and QPRAPIRPI (SEQ ID NO: 25).
The vaccines of the present invention may be used prophylactically or therapeutically.
The CTL epitopes may be synthesized using techniques well known to those skilled in this field. For example, the CTL epitopes may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled xe2x80x9cPeptide Synthesisxe2x80x9d by Atherton and Sheppard which is included in a publication entitled xe2x80x9cSynthetic Vaccinesxe2x80x9d edited by Nicholson and published by Blackwell Scientific Publications. Preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide (DMF). The polystyrene may be functionalised with chloromethyl or anionomethyl groups. Alternatively, cross-linked and functionalised polydimethyl-acrylamide gel is used which may be highly solvated and swollen by DMF and other dipolar aprolic solvents. Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads. In a preferred form, use may be made of commercial solid supports or resins which are selected from PALxe2x80x94PEG, PAKxe2x80x94PEG, KA, KR or TGR.
In solid state synthesis, use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the a-amino, carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive. Such functional groups can be selected from t-butyl esters of the structure RCOxe2x80x94OCMe3xe2x80x94COxe2x80x94NHR which are known as t-butoxy carboxyl or ROC derivatives. Use may also be made of the corresponding benzyl esters having the structure RCOxe2x80x94OCH2xe2x80x94C6H5 and urethanes having the structure C6H5CH2O COxe2x80x94NHR which are known as the benzyloxycarbonyl or Z-derivatives. Use may also be made of derivatives of fluorenyl methanol and especially the fluorenyl-methoxy carbonyl or Fmoc group. Each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made, for example, of BOC-benzyl and Fmoc-tertiary butyl protection strategies.
Reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides. This may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine. Activated esters such as those derived from p-nitrophenol and pentafluorophenyl may be used for this purpose. Their reactivity may be increased by addition of catalysts such as 1-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page 215-216 of the abovementioned Nicholson reference) also may be used. Other acylating species are formed in situ by treatment of the carboxylic acid (i.e. the Na-protected amino acid or peptide) with a condensing reagent and are reacted immediately with the amino component (the carboxy or C-protected amino acid or peptide). Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 of the Nicholson reference), O""Benzotriazole-N,N, Nxe2x80x2Nxe2x80x2-tetra methyl-uronium hexaflurophosphate (HBTU) and its analogous tetrafluroborate are frequently used condensing agents.
The attachment of the first amino acid to the solid phase support may be carried out using BOC-amino acids in any suitable manner. In one method BOC amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin. Fmoc-amino acids may be coupled to the p-alkoxybenzyl alcohol resin in similar manner. Alternatively, use may be made of various linkage agents or xe2x80x9chandlesxe2x80x9d to join the first amino acid to the resin. In this regard, p-hydroxymethyl phenylactic acid linked to aminomethyl polystyrene may be used for this purpose.
Details of the CTL epitopes of the present invention are set in Tables 3 and 4.
As will be readily appreciated by those skilled in the art the cytotoxic T-cell epitopes and vaccines of the present invention can be used to protect against EBV. Further given the possible greater involvement of type B EBV infection in immunocompromised individuals the present invention may have particular application in protection of individuals having decreased immune function, eg transplant patients.
In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following examples and Figures.